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Originally published In Press as doi:10.1074/jbc.M609822200 on November 30, 2006

J. Biol. Chem., Vol. 282, Issue 3, 1727-1737, January 19, 2007
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Translational Control of Glial Glutamate Transporter EAAT2 Expression*

Guilian Tian{ddagger}§, Liching Lai{ddagger}, Hong Guo{ddagger}, Yuan Lin{ddagger}, Matthew E. R. Butchbach{ddagger}§, Yueming Chang{ddagger}§, and Chien-liang Glenn Lin{ddagger}§1

From the {ddagger}Department of Neuroscience and §Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210

Received for publication, October 18, 2006 , and in revised form, November 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5'-untranslated regions (5'-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5'-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5'-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excitatory amino acid transporters (EAATs)2 in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Five sodium-dependent glutamate transporter subtypes have been identified and characterized: GLAST-1 (EAAT1) (1, 2), GLT-1 (EAAT2) (3, 4), EAAC1 (EAAT3) (5, 6), EAAT4 (7), and EAAT5 (8). EAAT2 is expressed mainly in glial cells throughout the brain and is responsible for up to 90% of all glutamate transport in adult tissue (9, 10).

A malfunction in the glutamate transport system can lead to accumulation of excessive glutamate in the synapse, which is harmful to neurons and could result in neurodegeneration (11, 12). Over the past 15 years, it has been demonstrated that glutamate-mediated toxicity may play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and Alzheimer disease (AD). Rothstein and colleagues (13) reported a significant increase in levels of glutamate in the cerebrospinal fluid of ALS patients and defective glutamate transport in the affected areas of ALS (14). Subsequent studies demonstrated a dramatic and selective loss of EAAT2 protein in the affected areas of ALS (15). Defective glutamate transport and loss of EAAT2 protein were also reported in the affected areas of AD patients (16, 17). This phenomenon also occurs in several rodent models of the diseases, including transgenic mice or rats expressing ALS-linked mutant superoxide dismutase (SOD1) (18–21) and transgenic mice expressing AD-linked mutant amyloid precursor protein (22). These rodent models demonstrated that down-regulation of EAAT2 protein occurs during the final stage of the pathology. We (23) modestly overexpressed EAAT2 in the SOD1G93A mouse to compensate for the loss of EAAT2 and demonstrated a delay in disease onset accompanied by a prolonged survival of motor neurons. In addition, Rothstein et al. (24) recently reported that increased expression of EAAT2 by ceftriaxone ameliorated symptoms and prolonged the survival of SOD1G93A mice. These studies imply that impairment of glutamate transport is a late contributing mechanism and not a major culprit for neuron degeneration.

The mechanism underlying the loss of EAAT2 is still unclear. It appears that this occurs due to disturbance at the post-transcriptional level, because EAAT2 mRNA is not decreased (15, 16, 25). In this study, we demonstrate that translation of EAAT2 mRNA is regulated by many factors. It is possible that reduced EAAT2 protein levels in ALS and AD may be partially due to lack of stimulating factors or the presence of inhibiting factors that repress translation of EAAT2 transcripts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Corticosterone, retinol, T3 (triodo-L-thyronine), biotin, vitamin B12, and cycloheximide were obtained from Sigma. B-27, B-27 without antioxidants, and N-2 were obtained from Invitrogen.

Identification of Initiation Sites—The 5' end of the EAAT2 transcript was analyzed using the RNA ligase-mediated rapid amplification of cDNA ends (5'-RLM-RACE) method according to the protocol provided with the FirstChoiceTM RLMRACE kit (Ambion, Austin, TX). Briefly, total RNA (10 µg) was dephosphorylated with 2 µl of calf intestinal phosphatase. The cap structure was subsequently removed with 2 µl of tobacco acid pyrophosphatase to produce a phosphorylated RNA at the 5' end, to which the 5'-RACE adapter (5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3') was ligated. The resulting RNA was reverse-transcribed using EAAT2 gene-specific primers (primer A1, 5'-GCTTGGGTTCCTCTGAGCCAAGATGACTGT-3', corresponding to position +59 downstream of ATG; primer B1, 5'-GGTGGCAGGAGCCCAGGATCTAAG-3', corresponding to position –771 upstream of ATG) and 200 units of SuperScript II reverse transcriptase. The resulting cDNAs were amplified by PCR using the RACE 5' outer primer (5'-GCTGATGGCGATGAATGAACACTG-3') and EAAT2-specific primers A1 and B1. The resulting PCR products were amplified further by nested PCR using the RACE 5' inner primer (5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3') and EAAT2-specific primers (A2, 5'-CCACCTGCTTGGGCATATTGTTGGCAC-3', corresponding to position +17 downstream of ATG; B2, 5'-CTATTGTTTCCCCTGAAGCCCGC-3', corresponding to position –800 upstream of ATG). The PCR conditions were as follows: 94 °C for 2 min, 85 °C for 2 min, followed by 10 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min and then 25 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min (increasing 0.5 s for each cycle). The reaction was then extended for 10 min at 72 °C. Nested PCR products were purified from 1% agarose gel and cloned using the PCR-ScriptTM Amp cloning kit (Stratagene, La Jolla, CA). Individual clones were sequenced for determination of the transcription start sites.

Abundance of EAAT2 mRNAs—Normal human frontal cortex mRNA was isolated from postmortem frozen tissues using TRIzol (Invitrogen) followed by Oligotex Suspension kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized with Thermoscript reverse transcriptase (Invitrogen) using primer A1 (described above). As a control EAAT2 RNA, an EAAT2 transcript that contains the 565-nucleotide (nt) 5'-UTR and 395 nt of coding region was generated by in vitro transcription using RiboMAXTM large scale RNA production systems (Promega, Madison, WI). The template for the generation of this control RNA was prepared from pcDNA3/EAAT2 by restriction digestion with ScaI (Invitrogen). The following primer combinations (indicated in Fig. 1C) were used for PCR: primers A1 + C (5'-CCCGGCGTCCGCTTTCTCCCT-3', corresponding to position –72 upstream of ATG), primers A1 + D (5'-CTGGGCGCATCGCTCTCTCG-3', –310 upstream of ATG), and primers E (5'-GGTAAGCCCTTTAGCGCCTC-3', –405 upstream of ATG) + F(5'-AAACCTTGCAATCCCTCCCTGGCCG-3', –525 upstream of ATG). The MasterTaq kit (Eppendorf, Westbury, NY), which is designed to increase reproducible yields from GC-rich templates, was used, and PCR conditions were as follows: 95 °C for 3 min, 85 °C for 2 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min for 30 cycles followed by 10 min of extension at 72 °C. The PCR products were resolved in 2% agarose gel, and the intensity of each band was analyzed by Kodak one-dimensional image analysis software (Eastman Kodak Co.).

Cell Cultures—Mouse primary cortical cultures were obtained as described previously (23). Briefly, cortices were dissected out of the newborn (P0–P1) brains and incubated in activated papain for 30 min at 37 °C, triturated by repeated pipetting with a small bore pipette and plated onto poly-D-lysine (0.1 mg/ml)-coated plastic culture dishes or glass slides. These cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 25 mM glucose, 1 mM sodium pyruvate, 19.4 µM pyridoxine hydrochloride, 2 mM glutamine, and 1% B-27 supplement (Invitrogen). Rat primary astrocyte cultures were obtained from Dr. Richard W. Burry (Department of Neuroscience, Ohio State University). The cells were maintained in DMEM with 10% fetal bovine serum (FBS) (Invitrogen). Human embryonic kidney cells (HEK293 cells) were cultured in DMEM with 10% FBS.

Generation of Plasmid DNA Constructs—To generate pcDNA3/EAAT2 with 76-nt 5'-UTR, DNA containing EAAT2 coding region and 76 bp of 5'-UTR was amplified by PCR using the primer set 5'-CCCGGCGTCCGCTTTCTCCCT-3' and 5'-GGATCCAGACTCATATCCTTATTTCTCACG-3' and pcDNA3/EAAT2 as template; this PCR product was inserted into pPCR-ScriptAmpSK(+) (Stratagene, La Jolla, CA) and then subcloned into pcDNA3 with EcoRI and NotI. pcDNA3/EAAT2 with a 310-nt 5'-UTR was generated in the same way as above except that the following PCR primers were used: 5'-CTGGGCGCATCGCTCTCTCG-3' and 5'-GGATCCAGACTCATATCCTTATTTCTCACG-3'. To generate pcDNA3/EAAT2 with a 1091-nt 5'-UTR, the 5'-UTR sequence from –1117 to –239 was subcloned from TOPO/EAAT2 promoter into pcDNA3/EAAT2 using SspI and SacII.

Generation of Stably Transfected Primary Astrocyte Cell Lines—Low passage (four passages) rat primary astrocyte cells were plated onto cell culture dishes and transfected with plasmid DNA (pcDNA3/EAAT2) using Lipofectamine Plus (Invitrogen) according to standard protocol. The medium was replaced with fresh medium containing Geneticin (0.9 mg/ml) 48 h post-transfection to select for EAAT2-expressing cells. Selection medium was replaced every 3 days until colonies formed (18–21 days later). Colonies were examined for EAAT2 expression by RT-PCR using EAAT2-specific primers and also by immunoblotting using a rabbit anti-EAAT2 pAb.

RT-PCR—Total RNA from primary astrocytes or primary cortical cultures was isolated with TRIzol (Invitrogen), and first strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Invitrogen) using an EAAT2-specific primer (5'-ACGCTGGGGAGTTTATTCAAGAAT-3'). beta-Actin was used as an internal control (primer, 5'-TGTCAAAGAAAGGGTGTAAAACGCAGC-3'). PCR primers 5'-GGCAACTGGGGATGTACA-3' and 5'-ACGCTGGGGAGTTTATTCAAGAAT-3' were used for EAAT2 cDNA, and primers 5'-CGGGACCTGACAGACTACCTCAT-3' and 5'-TGTCAAAGAAAGGGTGTAAAACGCAGC-3' were used for actin cDNA. PCR conditions were as follows: 95 °C for 3 min, 85 °C for 2 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min for 30 cycles followed by 10 min of extension at 72 °C.

Immunoblotting—Immunoblotting was performed as described previously (26). Briefly, the harvested samples were sonicated in PBS containing Complete protease inhibitor mixture (Roche Applied Science), assayed for protein concentration, resolved by SDS-PAGE (8% polyacrylamide), and transferred onto polyvinylidene difluoride membranes. The following primary antibodies were used: rabbit anti-EAAT2 pAb (1:4000), rabbit anti-EAAT1 pAb (1:200), rabbit anti-EAAT3 pAb (1:200), rabbit anti-glial fibrillary acid protein (GFAP) pAb (1:1000; Promega, Madison, WI), and goat anti-actin (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's directions. Band intensities were analyzed with Scion Image Release Beta 4.0.2 (Scion Corp.).

[3H]Glutamate Uptake Assay—Uptake of radiolabeled glutamate was monitored in cultured cells as described previously (27). Cultured cells grown on 6-well plates were washed with uptake sample buffer (320 mM sucrose in 50 mM Tris-HCl, pH 7.4) and then incubated for 10 min at 37 °C with L-[3H]glutamate (0.5 µCi; Amersham Biosciences) in either Na+-containing or Na+-free Kreb's buffer supplemented with 40 µM unlabeled glutamate. The cells were washed with ice-cold PBS and lysed in 1 mM NaOH. The amount of radiolabeled glutamate was measured using a Beckman Coulter LS6500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA). The amount of L-[3H]glutamate transported into the cells was calculated as previously described (27). Na+-dependent L-[3H]glutamate uptake was calculated by subtracting Na+-independent L-[3H]glutamate uptake (in Na+-free Kreb's buffer) from the total L-[3H]glutamate uptake (in Na+-containing Kreb's buffer). Protein concentrations were determined with the Coomassie Plus protein assay (Pierce). Na+-dependent L-[3H]glutamate uptake was expressed as nmol of L-[3H]glutamate/mg of protein/min.

Immunofluorescence—Fixation of cultured cells and immunofluorescent staining were accomplished as described previously (27). The following primary antibodies were used in this study: purified rabbit anti-EAAT2 pAb (1:200), purified rabbit anti-EAAT3 pAb (1:200), rabbit anti-GFAP pAb (1:1000; Promega, Madison, WI), and mouse anti-MAP2 monoclonal antibody (1:1000; Neomarker, Fremont, CA). Images were obtained using a Zeiss Axioskop 2 inverted microscope and AxioVision software.

Cell Surface Biotinylation—Labeling of proteins on the plasma membrane was accomplished by cell surface biotinylation as described previously (23). Cells were washed twice with PBS/CaMg (100 µM CaCl2 and 1 mM MgCl2 in PBS, pH 7.4) at room temperature and then incubated with biotinylation buffer (1 mg/ml EZ-Link® Sulfo-NHS-SS-Biotin (Pierce) in PBS/CaMg) for 20 min at 4 °C with constant shaking. Cells were then incubated with quenching buffer (100 mM glycine in PBS/CaMg) for 30 min at 4 °C with constant shaking and scraped in quenching buffer. The samples were sonicated in PBS containing Complete protease inhibitor mixture (Roche Applied Science) and lysed in 1% Triton X-100 (Roche Applied Science) for 1 h at 4 °C. Biotinylated proteins were recovered by incubation with 100 µl of immobilized NeutrAvidin (50% slurry; Pierce) at 4 °C overnight with end-over-end rotation. The avidin beads were recovered by centrifugation (12,000 x g for 5 min at 4 °C) and then washed four times with washing buffer (1% Triton X-100 in PBS containing Complete protease inhibitor mixture). After washing, the beads were resuspended in 1x SDS-PAGE loading dye.

Pulse-Chase Experiments—PA-EAAT2 cells were first cultured in 10% FBS for 24 h, washed twice with sterile PBS/CaMg (100 µM CaCl2 and 1 mM MgCl2 in PBS, pH 7.4) at room temperature, and then incubated with sterile biotinylation buffer (1 mg/ml EZ-Link® Sulfo-NHS-SS-Biotin (Pierce) in PBS/CaMg) for 20 min at 37 °C with gentle shaking every 5 min. Cells were then incubated with sterile quenching buffer (100 mM glycine in PBS/CaMg) for 30 min at 37 °C with gentle shaking every 5 min. After biotinylation, cells were cultured in DMEM with corticosterone (0.2 µg/ml) for 8 or 24 h and then harvested for biotin-labeled EAAT2 protein level analysis as described above.

In Vitro Translation—Each form of EAAT2 cDNA in pcDNA3 vector was used as a DNA template for TNT quick coupled transcription/translation systems in the presence of T7 RNA polymerase and [35S]methionine according to the manufacturer's protocols (Promega, Madison, WI). Equivalent copy numbers of plasmid DNAs were used in each assay. The reactions were carried out at 30 °C for 90 min. The synthesized protein products were electrophoretically resolved through an 8% SDS-polyacrylamide gel, and the gel was then dried and exposed to autoradiography film for 24 h.

Drug Administration—Intrathecal administration of drugs was performed as described previously by Hylden and Wilcox (28). Normal FVB mice (20–25 g) were used throughout these experiments. Briefly, a disposable 30-gauge one-half-inch needle (BD Biosciences) attached to a 50-µl Hamilton syringe (Hamilton, Reno, NV) was inserted into the intervertebral space between L5 and L6 level of the spinal cord. The accuracy of each injection was indicated by a characteristic reflexive flick of the tail. 1 µg/µl of corticosterone in 10% ethanol was injected in a volume of 5 µl/mouse, and 5 µl of 10% ethanol was injected into the control mice.

Statistical Analysis—The quantitative data in this study were expressed as the mean ± S.E. Statistical analysis was performed using the unpaired Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
There Are Multiple Transcriptional Initiation Sites of the EAAT2 Gene—Eukaryotic translation can be subdivided into three sequential phases of initiation, elongation, and termination. Frequently, it is the initiation phase that is targeted in translational regulation (29). Such regulation is mainly determined by structural properties of the mRNA primarily within the 5'-UTR. Su et al. (30) have reported that the major transcript of EAAT2 in primary human fetal astrocytes is initiated from an adenosine residue located 283 nt upstream of the ATG start codon. However, we found in a previous study (31) that some human EAAT2 transcripts contain at least 428 nt of 5'-UTR. In addition, a human EAAT2 cDNA that we obtained from another laboratory contained the 565-nt 5'-UTR. Furthermore, mouse EAAT2 transcripts contain at least 660 nt of 5'-UTR (32), and rat EAAT2 transcripts contain at least 621 nt of 5'-UTR (33). Su et al. (30) used primer extension analysis to determine the transcriptional initiation site. Shorter primer extension products can be generated if the primer target sequence is not close to the 5' terminus of the mRNA. This is due to the tendency of reverse transcriptase to stop or pause in a region of high secondary structure in the template RNA. We used the computer program MFOLD (available on the World Wide Web at bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) (34) to predict secondary structure of EAAT2 5'-UTR. There is a predicted region of strong secondary structure ({Delta}G = –99.3 kcal/mol) located from position –136 to –340 upstream of ATG. Moreover, we performed many RT-PCR experiments to amplify EAAT2 5'-UTR and learned that it is difficult to pass through the region around 250 nt upstream of ATG when using standard RT conditions.


Figure 1
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  		FIGURE 1.
Identification of the transcriptional initiation sites of the EAAT2 gene by 5' RLM-RACE analysis. A, agarose gel electrophoresis of nested PCR products from 5' RLM-RACE analysis. mRNA isolated from human frontal cortex was left untreated (lanes 3 and 5) or treated with tobacco acid pyrophosphatase (TAP)(lanes 2 and 4). Following RT and primary PCR reactions, nested PCRs were performed using 5' RACE inner primer/EAAT2 nested reverse primer A or primer B. B, EAAT2 5' RLM-RACE products were cloned into pPCR-Script and sequenced. Three initiation sites were identified. C, primer sets (C/A, D/A, and F/E) used to determine relative abundance of three forms of EAAT2 mRNAs by RT-PCR analysis. D, agarose gel electrophoresis of RT-PCR products. A control EAAT2 RNA was generated by in vitro transcription. Human mRNA (n = 3) was prepared from normal human frontal cortex. Serial dilutions of RT product were subjected to PCR using the indicated primer sets.

 
5' RLM-RACE is a reliable way to determine the transcriptional initiation site of the mRNA. Only authentic capped 5' ends of mRNA are detected by RLM-RACE. We first used the computer program Promoter 2.0 (www.cbs.dtu.dk/services/Promoter/) (35) to predict the potential transcriptional initiation sites. The result showed that there are three potential initiation sites within 1.2 kb upstream of ATG, each located at around –74, –345, and –1153 upstream of ATG. Primer A, which corresponds to position +59 downstream of ATG, was used to test the putative –74 and –345 initiation sites, whereas primer B, which corresponds to position –771 upstream of ATG, was used to test the putative –1153 initiation site. Tobacco acid pyrophosphatase-treated human frontal cortex mRNA was used as a template, since tobacco acid pyrophosphatase removes the cap structure from full-length mRNA, thereby leaving a free 5'-monophosphate to be ligated to an adapter for amplification. As shown in Fig. 1A, two PCR fragments were obtained when using primer A, and one PCR fragment was obtained when using primer B. No product was obtained when RNA was not treated with tobacco acid pyrophosphatase, indicating that these PCR fragments were derived from full-length capped mRNA. These PCR fragments were cloned and sequenced. They all contained adapter sequence and mapped the 5' end of the mRNA to 76, 310, and 1091 nt upstream of the ATG start codon (Fig. 1B). These results indicate that there are multiple transcriptional initiation sites of the EAAT2 gene.

What are the relative abundances of these three forms of EAAT2 mRNA in vivo? We initially attempted to answer this question by a ribonuclease protection assay but were not able to obtain reliable results, probably due to the strong secondary structure of the 5'-UTR. We then approached this question by using quantitative RT-PCR. mRNA samples from normal human front cortices (n = 3) and the control EAAT2 RNA, generated by in vitro transcription using EAAT2 cDNA as template, were subjected to RT. Primer A, which corresponds to position +59 downstream of ATG, was used for RT, which was performed at 60 °C using Thermoscript so as to easily pass through regions of high secondary structure. Serial dilutions of RT product were then subjected to PCR using primer sets indicated in Fig. 1C. The PCR product amplified by primer A and primer C represents all three forms of EAAT2 mRNA, whereas the PCR product amplified by primer A and primer D represents those mRNAs containing the 310- and 1091-nt 5' UTRs, and the PCR product amplified by primer E and primer F represents those mRNAs containing a 1091-nt 5'-UTR. PCR products were resolved by agarose gel electrophoresis (Fig. 1D). The intensity of each band was measured by densitometry and plotted as a function of the dilutions of the RT product from the control EAAT2 RNA, which was used to produce a standard curve for each set of primers. The copy number of each form of human EAAT2 mRNA was calculated from the appropriate standard curve, and the relative abundance of each form of EAAT2 mRNA was then determined by comparing the copy numbers among three forms. The results show that the proportion of the three forms of EAAT2 mRNAs with the 76-, 310-, and 1091-nt 5'-UTRs was ~45, 35, and 20%, respectively.

Translation of EAAT2 mRNA Is Regulated by Corticosterone and Retinol—The 5'-UTR of ~90% of vertebrate mRNAs are 10–200 nt in length, and those mRNAs that bear a considerably longer 5'-UTR (>200 bases) are often regulated at translational level (36). To test whether some of EAAT2 transcripts, especially those with 310- or 1091-nt 5'-UTRs, are translationally regulated, we generated a cell line that did not express endogenous EAAT2 but constantly expressed ectopic EAAT2 transcripts with long 5'-UTR. It is known that primary astrocyte-enriched cultures express very low or even undetectable EAAT2 (37–39). Several extracellular factors, such as epidermal growth factor (EGF), transforming growth factor {alpha}, platelet-derived growth factor, pituitary adenylate cyclase-activating polypeptide, and cAMP analogs have been identified to induce EAAT2 expression in primary astrocytes derived from cortical hemispheres but fail to affect EAAT2 expression in primary astrocytes derived from the cerebellum, mesencephalon, and spinal cord (40–42). We used primary astrocyte cultures derived from the cerebellum in this study and generated a stable line that constantly expressed a human EAAT2 transcript containing 565 nt of 5'-UTR and 79 nt of 3'-UTR, which was driven by the CMV promoter. This cell line does not express detectable endogenous EAAT2 even under EGF or dibutyryl-cAMP treatment but constantly expresses ectopic EAAT2 transcripts, which allows us to investigate whether translation of ectopic EAAT2 transcripts is regulated. We named this cell line PA-EAAT2.


Figure 2
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  		FIGURE 2.
EAAT2 protein expression is stimulated by many extracellular factors, including retinol and corticosterone. PA-EAAT2 cells were cultured in indicated medium conditions for 72 h (A–C) and then harvested for measuring EAAT2 protein levels by immunoblot analysis. 20 µg of total protein were loaded in each lane. A, FBS as well as the neural medium supplement B27 stimulated EAAT2 protein expression in PA-EAAT2 cells but not in the original primary astrocytes (PA). B, corticosterone (cortico) as well as retinol, which are components of B27 supplement, induced EAAT2 protein expression in PA-EAAT2. T3, triodo-L-thyronine; B27-antioxi, B27 minus antioxidants; Ethanol, vehicle. C, corticosterone- and retinol-induced EAAT2 protein expression in a dose-dependent manner. PA-EAAT2 was cultured in DMEM with the indicated dose of corticosterone or retinol. D, time course of corticosterone induction of EAAT2 protein expression. PA-EAAT2 cells were precultured in DMEM for 24 h and then treated with corticosterone (0.2 µg/ml) for the indicated time course. E, the translational control also occurs in HEK293 cells. Each experiment was repeated at least three times with consistent results.

 
PA-EAAT2 cells expressed low amounts of EAAT2 protein when they were cultured under serum-free medium (Fig. 2A, lane 3, DMEM); however, the EAAT2 protein level was significantly increased when the serum was added to the culture (Fig. 2A, lane 1, FBS). This induction was not observed in the original primary astrocytes (PA, Fig. 2A, lanes 7–12), indicating that the induced EAAT2 protein was produced from the CMV-driven EAAT2 cDNA. What are the factors in the serum that induce EAAT2 protein? We tested whether EGF can induce EAAT2 expression and found that EGF did not promote EAAT2 expression when PA-EAAT2 cells were cultured under serum-free medium (Fig. 2A, compare lane 3 (DMEM) and lane 4 (DMEM/EGF)), but it enhanced EAAT2 protein level when the serum was added to the culture (Fig. 2A, compare lane 1 (FBS) and lane 2 (FBS/EGF)). This result indicates that EGF needs other factors in the serum to have the effect. We then tested different neuron medium supplements, including B27 and N2, and found that B27 was able to induce EAAT2 expression (Fig. 2A, compare lane 3 (DMEM) and lane 6 (DMEM/B27)), but N2 did not induce EAAT2 expression (not shown). EGF further enhanced EAAT2 protein level when the presence of B27 (Fig. 2A, compare lane 5 (DMEM/EGF/B27) and lane 6 (DMEM/B27)). The B27 supplement consists of 32 components. We ruled out those components that are also present in N2 supplement and tested five potential components (corticosterone, retinol, T3 (triodo-L-thyronine), biotin, and vitamin B12) as well as B27 minus antioxidants (Fig. 2B, lanes 3–7). The results showed that corticosterone and retinol were able to independently increase EAAT2 protein levels (Fig. 2B, lane 3, DMEM/Corti, and lane 4, DMEM/Retinol), and their effects were additive (Fig. 2B, lane 9, DMEM/Corti/Retinol). These compounds increase EAAT2 protein levels in a dose-dependent manner (Fig. 2C). The stimulation immediately occurred within 1 h and was continuously enhanced with time (Fig. 2D). For this time course experiment, we precultured PA-EAAT2 cells in DMEM for 24 h and then treated the cells with corticosterone for different times in order to reduce the base line of EAAT2 protein levels so as to facilitate the detection of EAAT2 induction.

To examine if the observed translational control also occurs in other cell types, we generated a HEK293 cell line that constantly expressed ectopic EAAT2 transcripts containing 565 nt of 5'-UTR and 79 nt of 3'-UTR, which was driven by the CMV promoter. We found that the translation of this EAAT2 transcript was also regulated by corticosterone (Fig. 2E), indicating that the translational control is not specific to astrocytes.

To examine whether the induced EAAT2 was properly localized to plasma membrane, we performed cell surface biotinylation experiments. PA-EAAT2 cells were treated with corticosterone (0.2 µg/ml) or retinol (1.0 µg/ml) for 72 h and then incubated with Sulfo-NHS-SS-Biotin to label cell surface proteins followed by cell lysis. The resultant lysates were subjected to avidin affinity chromatography, and the avidin-bound proteins (B in Fig. 3A) and unbound proteins (flow-through (FT) in Fig. 3A) were analyzed for EAAT2 by immunoblotting. The proteins that were retained on the avidin beads are considered to be on the cell surface, whereas the unbound proteins are intracellular proteins. As shown in Fig. 3A, EAAT2 protein induced by corticosterone or retinol was present primarily in the cell surface fraction (B). Furthermore, we performed [3H]glutamate uptake assay to determine if the induced EAAT2 had glutamate uptake activity. As shown in Fig. 3B, [3H]glutamate uptake activity was significantly increased in corticosterone- or retinol-treated cells. These results indicate that corticosterone- or retinol-induced EAAT2 protein has normal cellular localization and glutamate transport function.


Figure 3
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  		FIGURE 3.
Corticosterone- or retinol-induced EAAT2 protein has normal cellular localization and glutamate transport function. PA-EAAT2 cells were cultured in DMEM and treated with corticosterone (cortico, 0.2 µg/ml) or retinol (1.0 µg/ml) for 72 h and then harvested for analyses. A, cell surface biotinylation was performed to separate cell surface proteins (avidin-bound) (B) from intracellular proteins (flow-through) (FT). Immunoblot analysis of both fractions revealed that the induced EAAT2 protein was properly localized to the cell surface. beta-Actin was served as a nonsurface protein control. B, [3H]glutamate uptake assay showed that Na+-dependent glutamate uptake activity was significantly increased in corticosterone- or retinol-treated cultures (*, p < 0.01). DHK (300 µM) was used to distinguish EAAT2-mediated glutamate uptake from that mediated by the other EAATs. Each experiment was repeated at least three times with consistent results.

 
What is the mechanism underlying the observed induction? It was not expected to be regulated at transcriptional level, because 1) the induction was not observed in the original primary astrocytes (Fig. 2A, lane 7–12, PA), indicating that corticosterone or retinol did not induce endogenous EAAT2 gene expression, and 2) the induction was not observed in a stable line expressing CMV promoter-driven luciferase (Fig. 4A), indicating that the induction by corticosterone or retinol was not a result of CMV promoter activation. To confirm this, we performed quantitative RT-PCR analysis to examine EAAT2 mRNA levels. This was done by running various numbers of PCR cycles and serial dilutions of RT product. The results showed that PA-EAAT2 cells expressed a high level of EAAT2 mRNA when they were cultured in DMEM, and the levels were not elevated in the corticosterone-, retinol-, or serum-added cultures (Fig. 4B shows the corticosterone result). To verify that our RT-PCR method could detect the difference between different amounts of RNA, we carried out RT reaction with different amounts of total RNA (2, 4, and 8 µg) isolated from PA-EAAT2 cells and PCR with serial dilutions of RT product using the same primer sets. Our method was able to efficiently detect these differences in RNA levels (data not shown). These results confirmed that the observed induction was not due to increased EAAT2 mRNA level.


Figure 4
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  		FIGURE 4.
Corticosterone as well as retinol stimulates translation of EAAT2 mRNA. A, primary astrocytes that stably express luciferase driven by the CMV promoter were cultured in DMEM and treated with corticoterone (Cortico) (0.2 µg/ml) or retinol (1.0 µg/ml) for 72 h and then harvested for immunoblot analysis using antibody against luciferase. 20 µg of total protein were loaded in each lane. Corticosterone as well as retinol did not induce luciferase expression. B, quantitative RT-PCR analysis. PA-EAAT2 cells were treated with corticosterone for 72 h in DMEM, and total RNA from these cells was prepared for analysis. beta-Actin RNA was served as an internal control. Corticosterone did not elevate EAAT2 mRNA level. C, pulse-chase experiments. PA-EAAT2 cells were preincubated with Sulfo-NHS-SS-Biotin to label surface EAAT2 proteins and then treated with corticosterone (0.2 µg/ml) in DMEM. Cells were harvested for measuring biotin-labeled EAAT2 protein levels by immunoblotting. Equal protein loading was confirmed by Ponceau S staining. Corticosterone did not prolong the lifetime of the EAAT2 protein. D, protein synthesis inhibition experiments. PA-EAAT2 cells were cultured in regular FBS medium for 24 h, preincubated with cycloheximide (cyclohex; 10 µM) in DMEM for 30 min and then treated with corticosterone (0.2 µg/ml) for 8 h. Cycloheximide prevented the corticosterone-mediated induction of EAAT2 protein. Each experiment was repeated at least three times with consistent results.

 
To test the possibility that corticosterone or retinol may prolong the stability of EAAT2 protein, we performed pulse-chase experiments. PA-EAAT2 cells were incubated with Sulfo-NHS-SS-Biotin for 20 min to label surface EAAT2 proteins with biotin and then treated with corticosterone (0.2 µg/ml). Cells were harvested at different time points and analyzed for biotin-labeled EAAT2 protein levels. We expected to see increased biotin-labeled EAAT2 protein in the corticosterone-treated cells over time if corticosterone prolonged the half-life of EAAT2 protein. The results showed that biotin-labeled EAAT2 protein levels were similar between treated and untreated cells (Fig. 4C), indicating that corticosterone did not increase the stability of EAAT2 protein. We also confirmed this result by treating cells with cycloheximide to inhibit the translation, and no induction of EAAT2 expression by corticosterone was observed under this condition (Fig. 4D). These results led us to conclude that the observed induction was regulated at the translational level.

5'-UTR of EAAT2 Transcripts Is Involved in the Translation Control—The initiation step is often the target of translational regulation. It is known that such regulation is mainly determined by structural properties of the mRNA primarily within the 5' UTR (43). In Fig. 1, we show that there are three EAAT2 mRNA transcripts that differ in the lengths of their 5'-UTRs. To test whether the translation of each form of EAAT2 transcript was regulated by corticosterone, we generated three primary astrocyte lines that constantly expressed EAAT2 transcripts containing 1091, 310, or 76 nt of 5'-UTR, respectively, followed by the coding region. These three stable lines were treated with corticosterone (in DMEM) for 72 h and then harvested for EAAT2 protein analysis. As shown in (Fig. 5A), corticosterone enhanced the translation of EAAT2 transcripts with 1091-nt and with 310-nt 5'-UTRs but not the transcript with 76-nt 5'-UTR. This result indicates that EAAT2 transcripts with long 5'-UTR are translationally regulated and that the region of the 5'-UTR between –76 to –310 is involved in this translational regulation.

We further confirmed the idea that EAAT2 5'-UTR is involved in translational control by in vitro translation. This was carried out in a coupled transcription/translation system using each form of EAAT2 cDNA in pcDNA3 vector. We made sure that equivalent copy numbers of plasmid DNA were used in these experiments. As shown in Fig. 5B, a significant amount of EAAT2 protein was generated from the cDNA containing short 5'-UTR (76 bp), but much less protein was produced from cDNAs with long 5'-UTR (310 or 1091 bp). To exclude the possibility that less EAAT2 RNA was generated from those cDNAs with long 5'-UTR using this coupled transcription/translation system thereby resulting in less protein production, we performed in vitro transcription separately and used equivalent amounts of EAAT2 RNA for in vitro translation. We obtained consistent results (not shown) with coupled transcription/translation reaction. These results suggest that the presence of long 5'-UTR significantly inhibits the translation of EAAT2, and this inhibition could be released by corticosterone.


Figure 5
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  		FIGURE 5.
5'-UTR of EAAT2 transcripts is involved in the translation control. A, three primary astrocyte lines that constantly expressed EAAT2 mRNAs containing either 1091, 310, or 76 nt of 5'-UTR were cultured in DMEM and treated with the corticosterone (0.2 µg/ml) for 72 h and harvested for immunoblot analysis using anti-EAAT2 antibodies. 20 µg of total protein were loaded in each lane. EAAT2 mRNAs containing 1091 or 310 nt of 5'-UTR are translationally regulated by corticosterone. B, in vitro translation of EAAT2. The equivalent amounts of EAAT2 cDNA (with either 76, 310, or 1091 bp of 5'-UTR) in pcDNA3 vector were subject to a coupled transcription/translation reaction in the presence of [35S]methionine. The translation of EAAT2 transcripts with long 5'-UTRs (310 or 1091 nt) was inhibited.

 


Figure 6
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  		FIGURE 6.
Translation of EAAT2 mRNA is regulated by many disease-associated insults. PA-EAAT2 cells were cultured in regular FBS medium, treated with the indicated factors for 72 h and harvested for immunoblot analysis using anti-EAAT2 antibodies. 20 µg of total protein were loaded in each lane. Glutamate (Glu)(A) and H2O2 (B) inhibited translation of EAAT2 mRNA. Amyloid beta peptide (Abeta 25–35) (C) and mutant SOD1G93A (D) enhanced the translation. Both penicillin (Pen) and ampicillin (Amp) induced the translation (E). Corticosterone was able to rescue the inhibition of EAAT2 expression by hydrogen peroxide and glutamate (F). Quantitative RT-PCR analysis revealed that EAAT2 mRNA levels remained constant after treatments (G). Con, control. Each experiment was repeated at least three times with consistent results.

 
Many Disease-associated Insults Affect the Efficiency of Translation of EAAT2 mRNA—In AD as well as in ALS, EAAT2 protein levels are decreased in the affected areas, whereas mRNA levels remain constant (15, 16, 25). Could loss of EAAT2 protein be partially due to translational misregulation in disease? To test this possibility, PA-EAAT2 cells were cultured in regular FBS medium, treated with different disease-associated insults for 72 h, and then harvested for EAAT2 protein levels. We tested four types of disease-associated insults. First, excess glutamate in extracellular space can lead to excitotoxicity from repetitive firing and/or elevation of intracellular calcium by calcium-permeable glutamate receptors, which has been long implicated in neuronal death (44). We found that glutamate inhibited translation of EAAT2 mRNA in a dose-dependent manner (Fig. 6A). Second, oxidative stress has been found to be part of pathogenesis of AD and ALS. Most cell culture models of oxidative stress employ hydrogen peroxide (H2O2) as the prooxidant to induce oxidative stress. We found that H2O2 significantly inhibited translation of EAAT2 mRNA (Fig. 6B). Third, amyloid beta peptide (Abeta) accumulation in senile plaques is a pathological hallmark of AD, and Abeta cascade is the most convincing hypothesis for the pathogenesis of AD. We found that a low dose of Abeta 25–35 (10–40 µM) enhanced translation of EAAT2 mRNA (Fig. 6C). This result is consistent with the previous report that a low dose of Abeta up-regulates EAAT2 expression in primary cortical astrocytes (45). Fourth, mutations in Cu2+/Zn+-superoxide dismutase (SOD1) are linked to familial ALS (46, 47). Transgenic mice that express mutant SOD1 develop similar clinical and pathological phenotypes to those seen in ALS (48–50). We found that expression of ALS-associated mutant SOD1G93A enhanced translation of EAAT2 mRNA (Fig. 6D). To confirm this result in vivo, we examined EAAT2 protein in the brains of SOD1G93A transgenic mice at presymptomatic stage (2 months old), and the result showed that the EAAT2 protein level was increased when compared with nontransgenic littermates (not shown). However, in the spinal cord of SOD1G93A mice at the symptomatic stage of disease (3 to 4 months old), the EAAT2 protein level is progressively decreased in the areas affected by neurodegeneration and reactive astrocytosis (19, 20).

Furthermore, Rothstein et al. (24) have recently reported that beta-lactam antibiotics are potent stimulators of EAAT2 expression. When used in SOD1G93A transgenic mice, the drug delayed loss of neurons and muscle strength and increased the average life span of these mice. We tested penicillin and ampicillin in PA-EAAT2 cells and found that both antibiotics were able to stimulate translation of EAAT2 mRNA (Fig. 6E).

We further tested whether corticosterone could rescue the inhibition of EAAT2 expression by H2O2 and glutamate. PA-EAAT2 cells were cultured in regular FBS medium, co-treated with corticosterone and glutamate or H2O2 for 72 h, and then harvested for EAAT2 protein levels. The results showed that the expression of EAAT2 in co-treated cells was higher than that in nontreated cells, indicating that corticosterone was able to rescue the inhibition (Fig. 6F).

In the above studies (Fig. 6, A–F), the cell number and the cell morphology were not visibly altered after treatments. Quantitative RT-PCR analysis showed that EAAT2 mRNA levels remained constant after treatments (Fig. 6G shows hydrogen peroxide, glutamate, and penicillin results). Moreover, we also tested these treatments in the primary astrocytes stably expressing CMV promoter-driven luciferase, and no induction or inhibition was observed (not shown).

Translational Regulation of EAAT2 mRNA Occurs in Primary Cortical Cultures and Mice—To examine whether the observed induction could occur in vivo, we first tested in the primary cortical cultures prepared from neonatal (P0–P1) normal mouse pups. The cultures primarily contained neurons that expressed EAAT3, type I astrocytes which expressed EAAT1 and type II astrocytes that expressed EAAT2 as determined by immunostaining and immunoblotting (not shown). The 7-day-old cultures were treated with corticosterone (0.1 and 0.2 µg/ml) or retinol (0.5 and 1.0 µg/ml) in serum-free conditions (DMEM) for 72 h and then harvested for analyses. Immunoblot analysis (Fig. 7A) showed that both corticosterone and retinol stimulated EAAT2 protein expression; however, the other glial glutamate transporter EAAT1 and neuronal glutamate transporter EAAT3 (not shown) were not affected by these two factors. Astrocyte marker GFAP was examined to indicate that there was no obvious difference in the number of astrocytes between treated and nontreated cells. Immunofluorescent staining (Fig. 7B) was also performed to confirm the results. GFAP and MAP2 (microtubule-associated protein 2) (neuron marker) staining showed that the number of astrocytes and neurons as well as their morphologies were not altered after treatments. Cell surface biotinylation experiments showed that the induced EAAT2 protein was primarily located at the cell surface fraction (Fig. 7C). Consistently, a [3H]glutamate uptake assay showed that glutamate uptake activity had an about 2-fold increase in corticosterone- or retinol-treated cultures (Fig. 7D). These results indicated that corticosterone and retinol specifically increased the level of functional EAAT2 protein. Furthermore, we examined EAAT2 mRNA levels by quantitative RT-PCR analysis, and the results showed that there was no difference between treated and nontreated cultures (Fig. 7E). Therefore, the enhancement in the expression of EAAT2 by corticosterone or retinol was due to the stimulation of translation.

Could corticosterone stimulate the expression of EAAT2 protein in the mouse? Corticosterone (1 µg/µl, 5 µl per injection) was administered intrathecally into the spinal cord of normal mice. After 3 days of treatment, animals (n = 7 per group) were sacrificed, and spinal cords were harvested for analyses. Corticosterone treatment led to more than 2-fold increase in EAAT2 protein level as determined by immunoblotting (Fig. 8, A and B). The other glutamate transporters EAAT1 (not shown) or EAAT3 were not changed after corticosterone administration. These primary culture and mouse studies indicate that translational regulation of EAAT2 mRNA occurs in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is known that expression of EAAT2 can be regulated by many extracellular factors, such as EGF, transforming growth factor {alpha}, platelet-derived growth factor, and pituitary adenylate cyclase-activating polypeptide, through increased transcription of the EAAT2 gene (41, 51, 52). In the present study, we demonstrate for the first time that expression of EAAT2 can be also regulated through increased translation of the EAAT2 transcript. We identified several factors, including corticosterone, retinol, and beta-lactam antibiotics, that can stimulate translation of EAAT2 transcript. We also found that many disease-associated insults affect the efficiency of translation of EAAT2, implicating that translational dysregulation may be involved in loss of EAAT2 in AD and ALS.

The human EAAT2 gene consists of 11 protein-coding exons with introns spanning more than 50 kb of genomic DNA on chromosome 11p13–12 (53). It encodes an open reading frame of 1722 nt, and multiple EAAT2 splice variants exist (31, 54–57). There are at least four 3'-variants of EAAT2 that originate from alternative cleavage of 3'-UTR (58). In the present study, we identified three distinct transcriptional initiation sites in human adult brains using 5' RLM-RACE, each located at –76, –310, and –1091 upstream of the ATG start codon (Fig. 1). The ratio of the three forms of EAAT2 mRNAs with the 76-, 310- and 1091-nt 5'-UTRs in human frontal cortex is ~45, 35, and 20%, respectively, as determined by quantitative RT-PCR method (Fig. 1). Alternative initiation sites may play an important role in differential EAAT2 protein expression under normal and disease conditions.

It is known that the mRNAs that bear long 5'-UTR (>200 nt) are often regulated at the translational level. We tested this idea initially in a primary astrocyte line stably expressing an EAAT2 transcript containing the 565-nt 5'-UTR and found that the rate of translation of this transcript was greatly enhanced by extracellular factors, including corticosterone and retinol (Fig. 2). We subsequently observed that corticosteroneor retinol-induced translation of EAAT2 also occurred in vivo, including primary cortical cultures (Fig. 7) and mice (Fig. 8). Many experiments were performed to solidly ensure that the observed induction was due to increased translation of EAAT2 and not because of increased transcription of the EAAT2 gene, increased stability of EAAT2 transcript, or increased stability of EAAT2 protein (Fig. 4).


Figure 7
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  		FIGURE 7.
Translational control of EAAT2 mRNA regulation occurs in the cortical primary neurons and astrocytes mixed cultures. The 7-day-old cortical primary dissociated cultures were treated with the indicated factors in serum-free condition (DMEM) for 72 h and then harvested for analyses. A, immunoblot analysis of total lysate prepared from the cultures showed that corticosterone (Cortico) and retinol-stimulated EAAT2 protein expression but not EAAT1 or GFAP. B, immunostaining of the cultures showed that EAAT2 immunoreactivity was significantly increased, and the number and the morphology of neurons/astrocytes were not altered in the treated cells. Scale bar, 50 µm. C, cell surface biotinylation was performed to separate cell surface proteins (avidin-bound) (B) from intracellular proteins (flow-through) (FT). Immunoblot analysis of both fractions revealed that the induced EAAT2 protein was properly localized to the cell surface. beta-Actin was served as a nonsurface protein control. D, the [3H]glutamate uptake assay showed that Na+-dependent glutamate uptake activity was significantly increased in corticosterone- or retinol-treated cultures (*, p < 0.01). DHK (300 µM) was used to distinguish EAAT2-mediated glutamate uptake from uptake mediated by the other EAATs. E, quantitative RT-PCR analysis revealed that mRNA levels are not elevated in the corticosterone- or retinol-treated cultures. Each experiment was repeated at least three times with consistent results.

 
What is the mechanism underlying the translational control of EAAT2 mRNA? EAAT2 transcripts with 1091- or 310-nt 5'-UTRs but not those with 76-nt 5'-UTRs are translationally regulated by corticosterone (Fig. 5), suggesting that the observed translational control probably occurs via cis elements in the 5'-UTR. The secondary structure analysis of EAAT2 5'-UTR revealed that there is a predicted region of strong secondary structure ({Delta}G =–99.3 kcal/mol) located from position –136 to –340 upstream of ATG. Highly structured 5'-UTR sequences tend to inhibit efficient translation. This inhibition is probably relieved by trans-regulatory factors that are stimulated by corticosterone, retinol, or beta-lactam antibiotics. The identification of the cis-regulatory elements and trans-regulatory factors involved in this regulation is currently under way.

It has been reported that a low dose of Abeta 25–35 (10–20 µM) (59) as well as Abeta 1–42 (1–5 µM) (45) enhances the clearance of extracellular glutamate mediated by increase of the expression of EAAT2 and EAAT1. However, a high dose of Abeta 25–35 (100 µM) inhibits Na+-dependent glutamate uptake, which can be prevented by antioxidants (60). Moreover, Landerback et al. (61) reported that high dose of Abeta 1–42 increases 4-hydroxy-2-nonenal conjugation the EAAT2, which could induce the oxidative damage of EAAT2. In the present study, we found that a low dose of Abeta 25–35 (10–40 µM) stimulated translation of the EAAT2 transcript (Fig. 6C). It is possible that at early stage of AD, a low dose of Abeta may induce the expression of EAAT2 and EAAT1, which leads to increased glutamate clearance by astrocytes. These effects may serve as a cellular defense against neurodegeneration in the pathogenesis of AD. However, at the end stage of AD, high concentration of Abeta may oxidatively damage EAAT2 protein, resulting in decrease of the EAAT2 protein level.


Figure 8
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  		FIGURE 8.
Corticosterone stimulates EAAT2 expression in mice. A, corticosterone (1 µg/µl) (Cortico) or vehicle (10% ethanol) was intrathecally injected into mouse spinal cord (5 µl/injection, n = 7 per group). The mice were injected once a day for 3 days, and the spinal cords were harvested for immunoblot analysis. Corticosterone-stimulated EAAT2 protein expression but not EAAT3 or actin. B, optical densities of EAAT2-specific bands were determined and normalized to actin (*, p < 0.05).

 
Corticosterone is a 21-carbon steroid hormone produced in the cortex of the adrenal glands. It is involved in the regulation of fuel metabolism, immune reactions, and stress responses. The retinol, the dietary form of vitamin A, is a fat soluble, antioxidant vitamin important in vision and bone growth. The action of corticosterone, retinol, and many other steroid hormones, is mediated primarily via specific, ligand-activated intracellular receptors (62). The binding of the steroid to the receptor exposes a nuclear localization signal, which targets the receptor complex to the nuclear compartment. Once in the nucleus, the receptor complex functions as a transcriptional activator or repressor modulating the expression of responsive genes. It has been reported that glucocorticoids acutely (within 4 h) repress protein synthesis in skeletal muscle probably via activation or repression of target genes that interfere with the initiation of translation (63). In the present study, we found that corticosterone as well as retinol was able to immediately stimulate translation of EAAT2 transcripts within 1 h (Fig. 2D). They probably activate or repress target factors, leading to enhancement of translation. The mechanisms underlying this stimulation process will be investigated in the future.

Consistent with our results, Zschocke et al. (42) have recently reported that the synthetic glucocorticoid dexamethasone and natural glucocorticoids could induce expression of the EAAT2 protein in astroglial cells derived from cortex. However, they showed that this induction was due to increased transcription of the EAAT2 gene. We examined EAAT2 mRNA levels in both PA-EAAT2 cells and primary cortical cultures treated with corticosterone, and mRNA levels were not elevated in the treated cells when compared with the nontreated cells. The difference between these two results is probably due to the cell type. In the Zschocke et al. (42) study, they used the primary cortical type I astrocytes, which is the major cell type in undifferentiated astrocyte monocultures expressing only the EAAT1 subtype (37, 38). In our experiments, we used primary cortical neuron and astrocyte mixed cultures. In this culture system, the astrocytes that are co-cultured with neurons develop a stellate morphology (differentiated type II astrocytes) and express EAAT2. The results from these two studies suggest that glucocorticoids could induce EAAT2 protein expression through both the transcriptional and translational level.

Rothstein et al. (24) recently reported that beta-lactam antibiotics, such as ceftriaxone, penicillin, and ampicillin can induce EAAT2 expression in both organotypic spinal cord slice cultures and mice. They believed that the regulation was mediated through increased transcription of the EAAT2 gene. We also observed that penicillin and ampicillin induced EAAT2 protein expression in PA-EAAT2 cells (Fig. 6F); however, our data indicated that this induction was due to increased translation of EAAT2 transcript. Rothstein et al. (24) did not measure the effect of beta-lactams on EAAT2 mRNA levels, so it is not clear whether the induction they observed was completely due to increased transcription of the EAAT2 gene. It is possible that beta-lactam antibiotics may regulate EAAT2 protein expression at both transcriptional and translation level. Further investigation of this regulation mechanism is needed.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant MH59805, the ALS Association, and the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Neuroscience, Ohio State University, 4198 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210. Tel.: 614-688-5433; Fax: 614-688-8742; E-mail: lin.492{at}osu.edu.

2 The abbreviations used are: EAAT, excitatory amino acid transporter; ALS, amyotrophic lateral sclerosis; AD, Alzheimer disease; SOD1, superoxide dismutase; RLM, RNA ligase-mediated; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); UTR, untranslated region; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcription; pAb, polyacrylamide; PBS, phosphate-buffered saline; GFAP, glial fibrillary acid protein; EGF, epidermal growth factor; CMV, cytomegalovirus. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey D. Rothstein for the glutamate transporter antibodies, Drs. Robert L. Stephens and Virginia M. Goettl for assistance with intrathecal injections, and Dr. Richard W. Burry for the reagents.



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 ABSTRACT
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 DISCUSSION
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